The present invention is directed to a chimeric gene that expresses in cotton cells insecticides having substantially the insect toxicity properties of the crystal protein produced by Bacillus thuringiensis.
Bacillus thuringiensis is a species of bacteria that produces a crystal protein, also referred to as delta-endotoxin. This crystal protein is, technically, a protoxin that is converted into a toxin upon being ingested by larvae of lepidopteran and dipteran insects.
The crystal protein from Bacillus thuringiensis is a potentially important insecticide having no known harmful effects on humans, other mammals, birds, fish or on insects other than the larvae of lepidopteran, coleopteran and dipteran insects. Other advantages of the use of the crystal protein from B. thuringiensis as an insecticide include its broad spectrum of activity against lepidopteran and dipteran insect larvae, and the apparent difficulty of such larvae to develop resistance against the crystal protein, even where the crystal protein is used on a large scale.
The crystal protein is effective as an insecticide when it is applied to plants subject to lepidopteran larvae infestation. Such plants include broccoli, lettuce and cotton. Lepidopteran larvae infestation is especially serious in cotton plants. Application of the crystal protein to plants has usually been accomplished by standard methods such as by dusting or spraying.
The use of the crystal protein as a commercial insecticide has, however, been inhibited by a number of disadvantages. For example, the protoxin remains on the surface of the plants being treated, where it is effective only against surface-feeding larvae, and where it is inactivated by prolonged exposure to ultraviolet radiation. This inactivation may be at least one cause of the general lack of persistance of the crystal protein in the environment. Accordingly, frequent and expensive application of the crystal protein is necessary.
By taking advantage of genetic engineering, a gene responsible for the production of a useful polypeptide can be transferred from a donor cell, in which the gene naturally occurs, to a host cell, in which the gene does not naturally occur; Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464. There are, in fact, few inherent limits to such transfers. Genes can be transferred between viruses, bacteria, plants and animals. In some cases, the transferred gene is functional, or can be made to be functional, in the host cell. When the host cell is a plant cell, whole plants can sometimes be regenerated from the cell.
Genes typically contain regions of DNA sequences including a promoter and a transcribed region. The transcribed region normally contains a 5' untranslated region, a coding sequence, and a 3' untranslated region.
The promoter contains the DNA sequence necessary for the initiation of transcription, during which the transcribed region is converted into mRNA. In eukaryotic cells, the promoter is believed to include a region recognized by RNA polymerase and a region which positions the RNA polymerase on the DNA for the initiation of transcription. This latter region, which is referred to as the TATA box, usually occurs about 30 nucleotides upstream from the site of transcription initiation.
Following the promoter region is a sequence that is transcribed into mRNA but is not translated into polypeptide. This sequence constitutes the so-called 5' untranslated region and is believed to contain sequences that are responsible for the initiation of translation, such as a ribosome binding site.
The coding region is the sequence that is just downstream from the 5' untranslated region in the DNA or the corresponding RNA. It is the coding region that is translated into polypeptides in accordance with the genetic code. B. thuringiensis, for example, has a gene with a coding sequence that translates into the amino acid sequence of the insecticidal crystal protein.
The coding region is followed by a sequence that is transcribed into mRNA, but is not translated into polypeptide. This sequence is called the 3' untranslated region and is believed to contain a signal that leads to the termination of transcription and, in eukaryotic mRNA, a signal that causes polyadenylation of the transcribed mRNA strand. Polyadenylation of the mRNA is believed to have processing and transportation functions.
Natural genes can be transferred in their entirety from a donor cell to a host cell. It is often preferable, however, to construct a gene containing the desired coding region with a promoter and, optionally, 5' and 3' untranslated regions that do not, in nature, exist in the same gene as the coding region. Such constructs are known as chimeric genes.
Genetic engineering methods have been described for improved ways of producing the crystal protein. For example, Schnepf et al, U.S. Pat. Nos. 4,448,885 and 4,467,036, describe plasmids for producing crystal protein in bacterial strains other than B. thuringiensis. These methods permit production of the crystal protein, but do not overcome the disadvantages of using the crystal protein as a commercial insecticide.
Suggestions have been made to clone B. thuringiensis toxin genes directly into plants in order to permit the plants to protect themselves; Klausner, A, Bio/Technology 2:408-419 (1984). Adang et al, European Patent Application 142, 924 (Agrigenetics), allege a method for cloning toxin genes from B. thuringiensis in tobacco and suggest protecting cotton the same way. Such a suggestion constitutes mere speculation, however, until methods for transforming cotton cells and regenerating plants from the cells are available. Such methods are described in U.S. patent application Ser. No. 122,200 filed Nov. 18, 1987 entitled "Regeneration and Transformation of Cotton", assigned to Phytogen, and U.S. patent application Ser. No. 122,162 filed Nov. 18, 1987 entitled "Regenerating Cotton from Cultured Cells", assigned to CIBA-GEIGY. U.S patent applications Ser. No. 122,200 and Ser. No. 122,162 were filed the same day as the present application. The disclosure of methods for transforming cotton cells in Phytogen patent application Ser. No. 122,200 and for regenerating cotton plants in Phytogen and CIBA-GEIGY patent applications Ser. No. 122,200 and Ser. No. 122,162 are incorporated herein be reference.
A need exists for developing new methods for producing the crystal protein of B. thuringiensis in cells of cotton plants and for new methods of killing lepidopteran larvae by feeding them cells of cotton plants containing a B. thuringiensis crystal protein or a similar polypeptide.